Cytotoxic, antioxidant, antibacterial activity of phytochemicals from Phragmanthera austroarabica

The cytotoxic, antioxidant, anticancer, and antibacterial properties of ethanolic extracts from Phragmanthera austroarabica is of interest. Plants of P. austroarabica were gathered from the southern Saudi Arabian region of Albaha. P. austroarabica extract was assessed using DPPH (2, 2-diphenyl-1-picrylhydrazyl). The German Collection of Microorganisms and Cell Cultures (DSMZ) cancer cell lines used in this investigation. The cytotoxic activity of P. austroarabica extract was explored against MCF-7 breast and A549 lung cancer cell lines, along with doxorubicin as a positive control. In both treated cells, P. austroarabica showed a remarkable activity via suppressing the cell's survival. In terms of IC50 (concentration equivalent to a survival rate of 50%), MCF-7 breast cancer cells were more sensitive to P. austroarabica extract.) DPPH colorimetric assay was employed to assess the antioxidant properties of P. austroarabica extract, the antioxidant activity was increased along with increment of extract concentrations. The leaves aqueous extract of P. austroarabica inhibited the growth of S. aureus by 6.3±0.12 mm and 24±0.43 mm and 15±0.56 mm respectively for seed, leaf and stem at concentrations 50 µl. However, the same concentrations inhibited the growth of E. coli by 25±0.75, 0.00 mm and 24±0.18 mm, following the same order. Different superscript letters indicate means that are significantly different at level (p<0.05). Minimal inhibitory concentrations (MIC) of P. austroarabica ethanolic extracts against the tested microorganisms were 1.5, 1.6 and 1.5, respectively for seed, leaf and stem against Staph. Aureus and were 1.2, 0.00 and 1.2, respectively for seed, leaf and stem against E. coli.


Background:
Biodiversity from nature, particularly plants, is still the important source of medicinal products since the past century [1].In the review by Newman and Cragg [2] it was stated that in the area of cancer drugs, from 155 small drugs molecules, 47% derived from or natural products itself.Therefore, exploration of new leads for drug discovery and development from plants is still important [3].For the country like Saudi Arabia, that is rich in plant biodiversity, opportunity to find new leads for drug discovery need to be explored by investigating the bioactivities of plants that already used for traditional/alternative medicine.Mistletoes are semi-parasitic perennial flowering plants that also known as medicinal plants [4].As a semi-parasitic plant, mistletoe is considered as an unwanted plant to economically important horticultural plant [5], however in the other side, mistletoe is known as one of medicinal plant used in traditional/alternative medicine several countries [6].According to [7], mistletoes are a representative of several families in the order Santalales, particularly Loranthaceae (1044 species) and Viscaceae (570 species).Nine mistletoe species from five genera and two families were found in the Kingdom of Saudi Arabia.Mistletoes have been used in folk medicine for a very long time in many different forms.Mistletoe preparations are widely used in many cultures on almost all continents to treat or manage a variety of medical problems such as hypertension, diabetes mellitus, inflammatory conditions, irregular menstruation, menopause, epilepsy, arthritis, cancer and etc. [11].Mistletoe preparations in the form of injectable extracts, infusions, tinctures, fluid extracts, or tea bags are widely used in many cultures on almost all continents [6].Lectins and viscotoxins, two of the most researched and potent phytocomponents of mistletoe, are crucial in the treatment of cancer because of their cytotoxic and apoptotic effects.The immunomodulatory effects of both groups have been demonstrated [12].The phenolic acids, phenylpropanoids, and flavonoids, which have antioxidant and anti-inflammatory properties and may reduce blood pressure, are another group of compounds found in mistletoe [13].
Triterpenic acids, in particular the cytotoxic and apoptotic oleanolic, ursolic, and betulinic acids, have also been discovered in mistletoe [14].Other significant pharmacological substances found in mistletoe include phytosterols, alkaloids, oligopeptides, polysaccharides, and fatty acids [15].Numerous variables relating to the environment and growing conditions, as well as the growth and development of the plant itself, have an impact on the accumulation of secondary metabolites in plants [16].It has been demonstrated in the past that the type of tree on which mistletoe grows affects its metabolic profile [17].Additionally, the biological activity of various plant parts can differ depending on the qualitative and quantitative composition of phytocomponents in those parts.It is necessary to clarify the differences between ethnomedical uses and modern pharmacology as well as between phytochemistry screening and structure elucidation.In-depth research should be done on the identification of bioactive compounds in crude extracts and fractions, the illustration of the underlying pharmacological mechanisms, as well as cytotoxicity, genotoxicity, and clinical trials of toxic tests [18].Therefore, it is of interest to evaluate the cytotoxic, antioxidant, anticancer and antimicrobial activities of water extracts of Mistletoe [19] collected from Saudi Arabia.

Plant material:
Phragmanthera austroarabica plants were collected from the Al-Baha region south-west of Saudi Arabia.The work carried out at Department of Biology Faculty of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), and Saudi Arabia.The plant materials (seeds, leaves and stems) were air-dried in shadow for 2 weeks then grounded into a powder and kept at room temperature (25ºC) until used.

Preparation of plant extract:
For ethanol extraction, 20 g of each of the air-dried powder was added to 100 ml of ethanol 70% and incubated for 24 hours on a shaker.Then it was filtered through 8 layers of muslin cloth and centrifuged at 5000 rpm for 10 minutes and the supernatant was collected.Then the concentrated to make the final volume onefourth of the original volume (stock solution).

Cytotoxic activity assessment of (MTT Assay):
The MTT assay was conducted following a previously described protocol (Nasr et al., 2020).In summary, cells in their exponential growth phase were trypsinized, counted, and seeded into 96well plates at a density of 50,000 cells per well in 100 μL of DMEM medium.After a 24-hour incubation period, the cells were exposed to varying concentrations of P. austroarabica extract (200, 100, 50, 25, and 0 μg/mL), with doxorubicin used as a positive control.Following 48-hour incubation, 10 μL of MTT solution (5 mg/mL in PBS) was added to each well and left for an additional 2-4 hours at 37°C.The resulting purple formazan product was dissolved using 100 μL of HCl isopropanol per well, and the plates were shaken for 10 minutes.The absorbance at 540 nm was measured using a plate reader (BioTek, USA).Dose-response curves were constructed to determine the IC50 (half-maximal concentration) values, which were calculated using OriginPro 8.5 software.The cell viability percentage was calculated as follows = [mean absorbance of the treated sample / mean absorbance of the control] × 100.

Cell culture:
The breast (MCF-7; ACC115) and lung (A549; ACC107) cancer cell lines utilized in this study were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), Braunschweig, Germany.The cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillinstreptomycin, which were procured from Gibco, Invitrogen, Thermo Fisher Scientific, and USA.

Antibacterial activities of plant extracts:
Antibacterial activity of each ethanolic extracts of P. austroarabica parts were determined by agar diffusion method.Fresh isolated colony of S. aureus and E. coli were suspended in sterile saline to get turbidity of 0.5 McFarland standards.A quantity of 0.1 ml of this suspension was spread aseptically on sterile Muller Hinton agar medium (Hi media).The wells of 6 mm diameter were bored by sterile cork borer.A quantity of 0.2 ml of each extract (100 mg/ml in 10% Dimethyl sulfoxide; DMSO was added to the wells.It was allowed to diffuse by keeping in freeze for 20 minutes.Ten percent (10%) DMSO in one of the wells was used as negative control.After diffusion of each extract, the plates were incubated at 37 °C for 24 hours.The inhibition zones were measured in mm.For each extract, three replicates were maintained.

Determination of minimum inhibitory concentration (MIC):
Tube dilution method was done to determine minimum inhibitory concentration of each extract.A series of two-fold dilutions of each extract ranging from 10 mg/ml S. aureus and E. coli to 0.3 mg/ml were made in Muller Hinton broth.A quantity of 0.1 ml of suspension of S. aureus and E. coli matched to 0.5 McFarland standard was seeded into each dilution.Two controls were maintained for each test batch.These controls included tube containing extract and growth medium without inoculum and organism control i.e., tube containing the growth medium and inoculum.The tubes were incubated at 37 °C for 24 hours and checked for turbidity.Minimum inhibitory concentration was determined as highest dilution of the extract that showed no visible growth.

Gas chromatography-mass spectrometry (GC-MS) measurement:
GC-MS analysis was performed using a Perkin Elmer Clarus 600 GC coupled with a mass spectrometer (Turbomass).A 1 µL extract volume was injected into the Elite5MS column, which possessed dimensions of 30 m in length, a film thickness of 0.25 µm, and an internal diameter of 0.25 µm.The injection was executed under the prescribed temperature protocol.The gas chromatography-mass spectrometry (GC-MS) system begins by setting the initial oven temperature at 40ºC and keeping it constant for 2 minutes.Following this, the temperature is increased to 200ºC with a pace of 5ºC per minute, and this heightened temperature is maintained for an additional duration of 2 minutes.Commencing at an initial temperature of 200ºC, the temperature exhibits a linear progression with a rate of 5ºC per minute, ultimately attaining a final value of 300ºC.Following this, the temperature remains consistent at this particular level for two minutes.The temperature of the injector was held constant at 280 ºC.The temperature of the interface was measured to be 240ºC, although the source's temperature was recorded as 220ºC.The system's vacuum pressure was maintained at a magnitude of 1.11 x 10^-5 torr, while the energy of the electrons was configured to be 70 electron volts (eV).In this experiment, helium was used as the mobile phase at a 1.0 mL/min flow rate.The mass spectra were obtained utilizing the electron ionization technique; with a scanning range from 40

Statistical analysis:
Data were recorded then plotted and statistically analyzed by using ANOVA one way to compare the mean± standard division of the tested sample.

Results: Cytotoxicity of P. austroarabica
The cytotoxic activity of P. austroarabica extract was explored against MCF-7 breast and A549 lung cancer cell lines, along with doxorubicin as a positive control.In both treated cells, P. austroarabica showed a remarkable activity via suppressing the cell's survival (Figure 1).In terms of IC50 (concentration equivalent to a survival rate of 50%), MCF-7 breast cancer cells were more sensitive to P. austroarabica extract (Table 1).Antioxidant properties of P. austroarabica: DPPH colorimetric assay was employed to assess the antioxidant properties of P. austroarabica extract.As depicted in (Figure 2), the antioxidant activity was increased along with increment of extract concentrations.Minimal inhibitory concentrations (MIC) of P. austroarabica ethanolic extracts against the tested microorganisms were 1.5, 1.6 and 1.5 respectively for seed, leaf and stem against Staph.Aureus and were 1.2, 0.00 and 1.2 respectively for seed, leaf and stem against E. coli shown Table 2.

Gas chromatography-mass spectrometry (GC-MS):
The chromatograms, of P. austroarabica ethanolic extracts obtained using gas chromatography-mass spectrometry (GC-MS), are depicted in Figure 1.The data exhibit discernible peaks, indicating the presence of 20 identifiable chemical compounds.Table 1 displays the compounds and their respective gas chromatography-mass spectrometry (GC-MS) information.
From the table, it can be seen that the percentage order of chemical compounds was as follows: Estragole   schimperi, P. curviflorus and T. globiferus are used to treat flatulence disease, to increase lactation and for removal of placenta in cow, camels and goats.Nevertheless, mistletoe rarely causes side effects when used in the recommended dosages.However, the side effects are more likely to occur when it is used in excessive doses.The side effects include headache, fatigue, chills, nausea, vomiting, upset stomach, fever, pruritus (itchy skin), and chills.These results in line with the reported cytotoxic activity of P. austroarabica extract against MCF-7 [28] and another breast cancer cells (MDA-MB-231) which may elucidate the cytotoxic properties of this species against breast cancer cells.
Moreover, earlier studies on Plicosepalus genus revealed several biological activities like antioxidant, antihepatotoxic, antidiabetic, antiviral, antimicrobial and cytotoxic activities [29].The use of T. globiferus as antifungal agent is potential as the methanol leaf extract and its fractions showed excellent antifungal activity against some selected fungal species including Candida albicans, Trychophyton mentagrophytes, Trychophyton rubrum and Aspergillus niger [30].In Saudi Arabia, O. glabratus aerial parts were studied, and some compounds that demonstrated cytotoxicity, antiviral activity against the hepatitis B virus, and anti-diabetic activity were isolated [31].The present investigation showed that the leaves aqueous extract of P. austroarabica inhibited the growth of Staph.aureus and E. coli.A variety of bacteria were used to test the activity of Viscum species in vitro [7].It was found that the extracts' antibacterial action was more potent toward gram-negative bacteria compared to gram-positive bacteria [32] [33].Viscum spp.'s antifungal activity was frequently examined on species of Candida, which are important microbes responsible for crucial morbidity as well as mortality in seriously ill hospitalized individuals [34].Nacsa-Farkas carried out a research experiment in which twelve species of Candida were tested, with Candida inconspicua being the most sensitive [35].Mistletoe's antiviral properties have not yet been thoroughly studied.The development of the human parainfluenza virus type 2 (HPIV-2) in vitro cells were found to be prevented by the water-based extract of leaves from Viscum album that develops upon lime trees.It was suggested that mistletoe might be helpful as an additional therapy for individuals with human immunodeficiency virus (HIV) because of its strong immune-boosting effects.In Saudi Arabia, the six different mistletoe species from four different genera of the family Loranthaceae that naturally grow in various locations were investigated for their antimicrobial activity by Waly [8].The antimicrobial effectiveness of the extract of mistletoe plant was evaluated against gram-positive (Staphylococcus aureus and Bacillus subtilis), gram-negative (Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi), and yeast (Candida albicans) bacterial and yeast strains.The antimicrobial findings indicated that, depending on the bacterial strain and the used concentration, methanolic extracts of the six Loranthaceae species showed varying degrees of growth inhibition.It was concluded that these plants of Plicosepalus, Phragmanthera, Tapinanthus and Oncocalyx should be taken in consideration, as reported in folk medicine, as potential.Three flavan derivatives, oncoglabrinol A, oncoglabrinol B, and oncoglabrinol C, were isolated from the active ethyl acetate extract of O. glabratus after chemical analysis of aerial parts collected from Saudi Arabia looked into the phytochemical components of the mistletoes of Saudi Arabia.The methanolic extracts' phytochemical screening revealed that flavonoids, steroids, and/or terpenoids were among their main constituents.Alkaloids, cardenolides, and saponins, on the other hand, were not found in any of the extracts that were examined.Anthraquinones and tannins are only weakly accumulated by P. austroarabica.The shoots of P. scurviflorus growing in Saudi Arabia were used to isolate the flavonoids catechin and quercetin, the naphthalene glycoside curviflorside, and the flavanol curviflorin [29].According to Abdallah et al. [48], Ocaffeoyl quinic acid conjugates and oléanane triterpenes were the main compounds that might be responsible for antihyperglycemic effect of V. schimperi.

Conclusion:
P. austroarabica showed a remarkable activity via suppressing the cell's survival.In terms of IC50 (concentration equivalent to a survival rate of 50%), MCF-7 breast cancer cells were more sensitive to P. austroarabica extract.)P. austroarabica has DPPH antioxidant properties.The antioxidant activity was increased along with increment of extract concentrations.P. austroarabica has antimicrobial activities against of S. aureus and E. coli.Different superscript letters indicate means that are significantly different at level (p˂0.05).Minimal inhibitory concentrations (MIC) of P. austroarabica ethanolic extracts against the tested microorganisms were 1.5, 1.6 and 1.5 respectively for seed, leaf and stem against Staph.Aureus and were 1.2, 0.00 and 1.2 respectively for seed, leaf and stem against E. coli.
to 600 m/z.Unidentified chemicals were discovered by comparing their spectra with those documented in the National Institute of Standard and Technology (2005) and WILEY (2006) libraries.The total time required to analyze a single sample was 58 minutes.

Figure 1 :
Figure 1: Cytotoxicity of P. austroarabica extract on human breast MCF-7 and lung A549 cancer cell lines assessed by using MTT assay.Values represent AVG ± SD of three independent experiments.

Figure 2 :
Figure 2: Antioxidant potential of P. austroarabica extract using DPPH assay.Values represent % radical scavenging (AVG ± SD) of three replicates.Antibacterial activity: Table 2, Figures3 & 4shows the leaves aqueous extract of P. austroarabica inhibited the growth of Staph.aureus by 6.3±0.12mm and 24±0.43mm,15±0.56 respectively for seed, leaf and stem at concentrations 50 µl.However, the same concentrations inhibited the growth of E. coli by 25±0.75, 0.00 mm and 24±0.18mm, following the same order.Different superscript letters indicate means that are significantly different at level (p˂0.05).Minimal inhibitory concentrations (MIC) of P. austroarabica ethanolic extracts against the tested microorganisms were 1.5, 1.6 and 1.5 respectively for seed, leaf and stem against Staph.Aureus and were 1.2, 0.00 and 1.2 respectively for seed, leaf and stem against E. coli shown Table2.

Figure 3 :
Figure 3: Antibacterial activity (mm) of P. austroarabica ethanolic extracts.A and D: Seed extract; B and E: leaf extract; C and F: stem extract.

Table 1 :
The IC50 values of P. austroarabica in various cancer cells

Table 3 :
Minimal inhibitory concentrations (MIC) of P. austroarabica ethanolic extracts against the tested microorganisms

Table 4 :
GC-MS information of P. austroarabica extract [25,26]P.curviflorus, which was collected in Saudi Arabia, may hold promise for the creation of prostate cancer chemotherapeutics[25,26].In addition, [27] performed a study to identify and catalog the medicinal plants used in the traditional system in various regions of Saudi Arabia to treat various livestock ailments.The study concluded that O.

38][36][37].
[40]rding the mistletoe pharmacological value, over decades, various preparations of mistletoe extract developed, including aqueous, hydroalcoholic, and ethanolic extracts [When using whole extracts rather than just purified lectins and toxins, the observed pharmaceutical effects are typically easier to identify [38].Its therapeutic effects have been shown to be cytotoxic, an apoptosis inducer, and an immunomodulator.In a rodent model, Parvez and Rishi confirmed the hypoglycemic salutation of O. glabratus and showed that its newly isolated flavan derivative Oncoglabrinol C activated PPARα/γ in liver cells (HepG2)[39].The ability of Oncoglabrinol C to reverse endothelial oxidative and apoptotic damage as well as to activate hepatic CYP3A4 was demonstrated.This justifies additional research into Oncoglabrinol C and related compounds in order to create efficient and secure medications for cardiovascular disorders linked to diabetes[40].The shoots of P. scurviflorus growing in Saudi Arabia were used to isolate naphthalene, the flavanol curviflorin, catechin, and quercetin.There have been reports of naphthalenes in liverworts, plants, insects, and fungi.According to Ibrahim and Mohamed (2017), naphthalenes have a variety of bioactivities, including antimicrobial, antioxidant, cytotoxic, anti-inflammatory, antiplatelet aggregation, and antiprotozoal.There are several medications that contain naphthalene, including nafacillin, naftifine, tolnaftate, and terbinafine, which are essential for managing microbial infection [41].From various Plicosepalus species, flavonoids, phenolic acids, triterpenes, sterols, and sesquiterpene lactones have been isolated.Plicosepalus curviflorus has been used to isolate flavane gallates, triterpenes, and sterols [45, 46].Alkaloids, flavonoids, polyphenols, and tannins are among the bioactive metabolites found in the aqueous extracts of the entire T. globiferus plant [47].